Regular exercise has proven to be a powerful and cost-effective intervention for the treatment and secondary prevention of coronary artery disease. However, detailed understanding of the fundamental cellular and molecular mechanisms that underlie exercise-induced cardioprotection remains incomplete, limiting the development of effective new therapeutic strategies for diseased patients. The distinction of reactive oxygen species (ROS) as contributors to pathophysiology of the vascular system is well recognized and targeting of ROS is regarded as a promising therapeutic approach for vascular disease states. Contrary to this characterization, our recent studies have revealed that exercise training produces significant physiologic adaptations in ROS signaling that reverse impaired vasodilation in collateral-dependent arterioles that are distal to chronic coronary artery occlusion. These novel findings support our central hypothesis that ROS play a critical protective role in the exercise training-induced restoration of vasodilation responses of the coronary microcirculation and thereby enhance perfusion and contractile function of the myocardium at risk. We propose that identification of the cellular sources of protective ROS, as well as delineation of downstream signaling pathways that promote restoration of myocardial perfusion will establish a valuable foundation for development of effective therapeutic strategies for coronary artery disease. Our four Specific Aims will test the following hypotheses: 1a) exercise training increases vascular NADPH oxidase (NOX)-mediated production of hydrogen peroxide (H2O2), independent of changes in superoxide anion production, under both basal and agonist- stimulated conditions in arterioles from chronically occluded hearts; 1b) production of H2O2 by the NOX4 isoform will be the primary mechanism that contributes to this exercise training-induced adaptation; 2) enhanced endothelium-dependent dilation after exercise training is attributable to an increased contribution of endothelial NOX4 isoform via increased protein levels of NOX4 or associated cofactors or a change in the subcellular localization of these molecules; 3a) H2O2 mediates vasodilatory effects via direct PKG1a dimerization with subsequent myosin light chain dephosphorylation and increases in large conductance, calcium-dependent K+ (BKCa) channel activity; 3b) exercise training increases activity of this pathway via increases in H2O2 production or increased colocalization of pathway proteins; 4) exercise training increases the contribution of vascular H2O2 and BKCa channels to augment regional myocardial perfusion and subsequently enhance cardiac contractile function both at rest and during increased cardiac workload in chronically occluded hearts. These studies will provide new insights into protective adaptations of reactive oxidants with exercise training which may emerge as important factors and therapeutic targets in managing cardiovascular disease.